This invention relates to methods for the production of the antibiotic, clavulanic acid.
Clavulanic acid is a broad spectrum beta-lactamase inhibitor and is an important antibiotic for the treatment of infectious diseases. It is produced commercially by the gram-positive mycelial prokaryote Streptomyces clavuligerus, which also produces the xcex2-lactam antibiotics penicillin N, desacetoxy cephalosphorin C and cephamycin C. Until recently, however, the pathway employed for clavulanic acid biosynthesis was much less well understood than the pathways leading to these other antibiotics.
Without knowledge of the pathway for clavulanic acid biosynthesis, it was not possible to isolate the genes coding for the key enzymes and to manipulate these genes to increase antibiotic yield or permit production of the antibiotic in heterologous systems.
One of the earliest enzymes of the pathway to be purified and characterised was clavaminic acid synthase. Two isozymes have now been identified and characterised (Marsh et al., (1992), Biochem., vol. 31, pp. 12648-657).
European Patent Application 0349121 describes a 6.8 kb Bgl II restriction fragment isolated from S. clavuligerus that encodes a portion of the genetic information involved in clavulanic acid synthesis. No further characterization of this fragment was performed nor was the utility of this fragment determined.
Marsh et al (1992, Biochem. vol 31, pp. 12648-657) cloned and sequenced two isozymes of clavaminate synthase cs1 and cs2 separated by 28 kb, however their location relative to the cephamycin and penicillin biosynthetic clusters was not determined. Ward and Hodgson (1993, FEMS Microbiol. Lett. vol 110, pp. 239-242) reported on the occurrence of a biosynthetic gene cluster for clavulanic acid. Aidoo et al (1994, Gene vol 147, pp. 41-46) cloned and sequenced cla the gene encoding a protein (possibly proclavaminic acid amidinohydrolase) involved in clavulanic acid production. They reported that this gene was upstream from and adjacent to an ORF with the identical sequence of cs2. Paradkar and Jensen (1995, J Bacteriol, vol 177, pp. 1307-1314) further analyzed a 6.6 kb Bgl II fragment from S. clavuligerus comprising cs2 and through gene disruption experiments demonstrated its role in clavulanic acid synthesis.
Until the work of the present inventors, the complete complement of genes required for clavulanic acid synthesis had not been identified.
The present inventors have now isolated, cloned and sequenced a 15 kb DNA fragment from S. clavuligerus which encodes 10 ORFs. Within this 15 kb of DNA lies an 11.6 kb Eco RI fragment which codes for eight proteins and enables the production of clavulanic acid by transformants of non-clavulanic-producing organisms. This 11.6 kb fragment includes 8 complete ORFs (ORFs 2 to 9), two of which have been previously characterized (Marsh et al 1992, Paradkar and Jensen 1995). ORF1, which is incomplete, is not involved in clavulanic acid synthesis, ORF4 encodes the CLA protein and ORF5 is cs2, encoding one of the isozymes of clavaminate synthase. The function of the other remaining ORFs within this 11.6 kb fragment or their role in clavulanic acid synthesis is unknown.
An isolated genomic DNA molecule of 15 kb is provided comprising the nucleotide sequence set out in SEQ ID NO:1. This DNA molecule comprises 10 ORFs, eight of which are involved in clavulanic acid synthesis. A process is provided for producing clavulanic acid in a transformant of a non-clavulanate-producing host.
The present invention provides isolated DNA molecules comprising the nucleotide sequence of SEQ ID NO: 15, 16, and 19 to 22.
Furthermore, the present invention is directed to DNA molecules comprising the nucleotide sequences that encode the amino acid sequence found in SEQ ID NO""s: 4, 5, and 8 to 11.
This invention also embraces DNA molecules comprising a nucleotide sequence of ORFs 2, 3, 6, 7, 8 and 9 and their associated non-transcribed regions. Such isolated DNA molecules have the nucleotide sequences found in SEQ ID NO""s: 15, 16 and 19 to 22.
This invention also embraces DNA molecules comprising the nucleotide sequences encoding the amino acid sequences of SEQ ID NO:""s 4, 5, and 8 to 11. These amino acid sequences correspond to the expression products of ORFs 2, 3, 6, 7, 8 and 9 as defined in SEQ ID NO""s: 4, 5 and 8 to 11.
This invention is also directed to recombinant vectors that comprise DNA molecules as defined above where hosts that have been transformed with these recombinant vectors.
This invention is also directed to the process for producing clavulanic acid in a non-clavulantic acid producing host that comprises transforming the host with a DNA molecule as defined above and culturing the host under suitable conditions to produce clavulanic acid.
Furthermore, this invention is directed to processes for enhancing clavulanic acid production in a clavulanic acid producing host comprising transforming the host with a DNA molecule as defined above.